Species delimitation for Neonectria ... - Micologia - UCRmicologia.biologia.ucr.ac.cr/PDFs/Neococcinea.pdf · Yuuri HIROOKA et al. / Species delimitation for Neonectria coccinea group

菌物学报 [email protected] 15 May 2013, 32(3): 485-517 Http://journals.im.ac.cn Mycosystema ISSN1672-6472 CN11-5180/Q © 2013 IMCAS, all rights reserved.

*Corresponding author. E-mail: [email protected] Received: 18-12-2012, accepted: 13-03-2013

Species delimitation for Neonectria coccinea group including the causal agents of beech bark disease in Asia, Europe, and North America Yuuri HIROOKA1* Amy Y. ROSSMAN2 Wen-Ying ZHUANG3 Catalina SALGADO-SALAZAR4 Priscila CHAVERRI4

1Forestry & Forest Products Research Institute, Department of Forest Microbiology, 1 Matsunosato, Tukuba, Ibaraki, 305-8687, JAPAN 2Systematic Mycology and Microbiology Laboratory, United States Department of Agriculture, Agricultural Research Service, 10300

Beltsville Ave., Beltsville, Maryland 20705, USA 3State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China 4University of Maryland, Department of Plant Science and Landscape Architecture, 2112 Plant Sciences Building, College Park, Maryland

20742, USA

Abstract: Neonectria coccinea has historically been known as a serious pathogen of beech trees in North America and

Europe. Phylogenetic relationships of this species and its relatives have been determined. Neonectria faginata, previously

referred to as Neo. coccinea var. faginata has been delimited from Neo. coccinea sensu stricto and Neo. punicea. Five

additional lineages supported by relatively high phylogenetic values are allied with Neo. coccinea and its relatives. Until

now, these lineages have not been recognized as species because no significant morphological differences among them were

found. Although each phylogenetic species appeared to be unique in host specificity and geography, relatively few specimens

of these species existed. We re-evaluated these species using newly obtained specimens and isolates from Asia, Europe, and

North America. Based on sequences of multiple loci, specifically act, ITS, LSU, rpb1, tef1 and tub, our results indicate that

these isolates represent four phylogenetic species, Neo. coccinea, Neo. faginata, Neo. microconidia, and Neo. punicea. In our

phylogeny, each species is well supported by high BI PP, MP BP, and ML BP values. The single-locus analyses of ITS and

LSU did not correlate with our species recognition criteria. Based on morphology, phylogeny, host specificity and

geographical traits, these four species are conclusively delimited. Each of them is re-described and illustrated. Epitype

specimens for Sphaeria coccinea and Neonectria coccinea var. faginata are designated. Because no type specimen of Neo.

punicea was found, this name is neotypified. Within Neo. coccinea, Neo. microconidia, and Neo. punicea, some sublineages

were supported by high phylogenetic values. In the future these may warrant recognition as distinct species.

Key words: invasive pathogens, molecular systematics, Neonectria coccinea, Neonectria faginata, Neonectria microconidia,

Neonectria punicea, species concept, typification

ISSN1672-6472 CN11-5180/Q Mycosystema May 15, 2013 Vol.32 No.3

http://journals.im.ac.cn/jwxtcn

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含亚洲、欧洲、北美洲山毛榉病害病原菌的 Neonectria coccinea 复合群的物种概念 Yuuri HIROOKA1* Amy Y. ROSSMAN2 Wen-Ying ZHUANG3 Catalina SALGADO-SALAZAR4 Priscila CHAVERRI4 1Forestry & Forest Products Research Institute, Department of Forest Microbiology, 1 Matsunosato, Tukuba,

Ibaraki, 305-8687, JAPAN [日本] 2Systematic Mycology and Microbiology Laboratory, United States Department of Agriculture, Agricultural

Research Service, 10300 Beltsville Ave., Beltsville, Maryland 20705, USA [美国] 3State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101,

P.R. China [中国] 4University of Maryland, Department of Plant Science and Landscape Architecture, 2112 Plant Sciences

Building, College Park, Maryland 20742, USA [美国]

摘要：对包含亚洲、欧洲、北美洲山毛榉病害病原菌在内的 Neonectria coccinea 复合群中物种之间的系统演化关系

以及物种概念进行了探讨。Neonectria faginata（曾处理为 Neo. coccinea var. faginata）与狭义的 Neo. coccinea 和 Neo.

punicea 有明显区别。系统树显示 5 个分支与 Neo. coccinea 群的关系较为接近，由于缺乏显著的形态差异，它们始终

没有在物种的等级上区分。根据形态学、系统发育、寄主专化性和地理因素，采用多基因 act、ITS、LSU、rpb1、tef1

和 tub 序列分析的方法，对亚洲、欧洲、北美洲的相关材料进行了研究，结果表明，它们代表 4 个独立的物种，即

Neo. coccinea，Neo. faginata，Neo. microconidia 和 Neo. punicea，对每个种进行了详尽的描述和图示。并为 Sphaeria

coccinea 和 Neonectria coccinea var. faginata 指定了附加模式。

关键词：入侵性病原菌，分子系统学，猩红新丛赤壳，Neonectria faginata，小孢新丛赤壳，Neonectria punicea，物种

概念，选模式

INTRODUCTION The beech bark disease (BBD) constitutes one

of the greatest global threats to beech forests. This disease occurs following the interaction between the beech scale insect, Cryptococcus fagisuga Lind. and one of the three ascomycetous fungi, Neonectria coccinea (Pers.) Rossman & Samuels, Neo. faginata (M.L. Lohman, A.M.J. Watson & Ayers) Castl. & Rossman, and Neo. ditissima (Tul. & C. Tul.) Samuels & Rossman. The disease was first documented in Germany in 1849 (McIntosh 1849).

The beech scale insect was introduced into Nova Scotia through seedling beech trees brought from Europe in 1890 (Ehrlich 1934; Hewitt 1914). Recent molecular studies indicate that the causal agents of BBD were introduced from Europe, yet it is unclear if the beech scale insect and BBD fungal pathogens were introduced into North America at the same time (Mahoney et al. 1999; Gwiazdowski et al. 2006). The disease has now rapidly spread across the northeastern United States and southeastern Canada (Houston 1994; Griffin et al. 2003) with

Yuuri HIROOKA et al. / Species delimitation for Neonectria coccinea group including the causal agents of beech bark disease in Asia, …

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80%–90% mortality rates of mature beech (Houston 1994).

The BBD pathogenic fungi are Neonectria coccinea on Fagus in Europe, Neo. faginata on Fagus in North America, and Neo. ditissima on hardwoods in North America and Europe, rarely Asia. These fungi have red, globose, slightly roughened perithecia, 1-septate ascospores, and cylindrocarpon-like anamorphs (Rossman et al. 1999). They are morphologically similar and often misidentified because they are distinguished by only small differences in ascospore size. Castlebury et al. (2006) conducted taxonomic studies of these fungi based on morphological observation and combined phylogenetic analyses of rpb2, tef1, and tub gene regions. They clarified the differences between Neonectria coccinea, Neo. ditissima, and Neo. faginata (=Neo. coccinea var. faginata), newly recognized at the species rank. The phylogenetic tree of Castlebury et al. (2006) also showed that potentially five distinct species exist that are sister to Neo. coccinea. Although each phylogenetic species seemed to be segregated by host specificity and geography, no significant morphological differences among them were found due to the limited number of samples.

Neonectria coccinea was originally described as Sphaeria coccinea Pers.: Fr. by Persoon (1800). After Fries (1823) sanctioned Sphaeria coccinea, he transferred this species to Nectria (Fries 1849). Because Persoon (1800) and Fries (1823, 1849) did not typify any specimens, Booth (1959) found several Persoon specimens of S. coccinea and designated one of them as the lectotype. Since the 19th century, the broadly conceived genus Nectria has been narrowed. Most recently the genus Nectria was determined to include fungi having red perithecia without bright yellow scurf and

tubercularia-like anamorph (Hirooka et al. 2011, 2012). Previously considered to be a synonym of Nectria, Rossman et al. (1999) resurrected the genus Neonectria Wollenw. (Wollenweber 1916), typified by Neo. ramulariae Wollenw., for nectria-like species having a cylindrocarpon-like anamorph. Many nectrioid species having cylindrocarpon-like anamorphs were included in the genus Neonectria. Chaverri et al. (2011) divided Neonectria into five genera: Campylocarpon Halleen, Schroers & Crous, Ilyonectria P. Chaverri & C. Salgado, Neonectria Wollenw., Rugonectria P. Chaverri & Samuels, and Thelonectria P. Chaverri & C. Salgado. These genera were defined based on a multiple locus phylogeny and morphological observations.

Neonectria coccinea with its anamorph Cylindrocarpon candidum (Link: Fr.) Wollenw. has been considered a common species distributed widely in the northern hemisphere, frequently reported on twigs and wood of beech trees as well as on other deciduous trees, dung, and soil (Domsch et al. 2007). The basionym of the anamorphic fungus, Fusidium candidum Link, was described by Link (1809) and sanctioned by Fries (1832). Wollenweber (1928) transferred Fusidium candidum to Cylindrocarpon. Booth (1966) first introduced the teleomorph-anamorph connection as states of one species, and recent studies have confirmed the connection. The recent study by Castlebury et al. (2006) restricted this species to Fagus in Europe.

Using combined sequences of several loci, a number of new species within Neonectria sensu lato were recently described from China (Luo & Zhuang 2010; Zhao et al. 2011). Neonectria confusa J. Luo & W.Y. Zhuang was placed in the Neo. coccinea group while Neo. microconidia J. Luo, P. Zhao & W.Y. Zhuang was sister to the Neo. coccinea clade. Although Luo & Zhuang (2010) and Zhao et al.



488

(2011) recognized Neo. confusa and Neo. microconidia as distinct species, the variation between them was relatively low with subtle morphological differences from other species in the Neo. coccinea group. A taxonomic reappraisal of the Neo. coccinea group is needed.

To determine the species circumscription within the Neo. coccinea group, detailed morphological and molecular phylogenetic analyses were undertaken. Many isolates from freshly collected and herbarium specimens around the northern hemisphere, particularly Asia, Europe, and North America, were used to define the species in the Neo. coccinea group. In order to identify the most suitable locus for recognition of cryptic species in this group, the multi-locus genealogies as well as a single locus approach were compared. Each species is described and illustrated, and a key is provided.

1 MATERIALS AND METHODS Specimens and isolates

Specimens and isolates of the Neo. coccinea group preserved in herbaria and culture collections were obtained and examined in this study. Fresh specimens of the teleomorphs were collected and sent by authors or collaborators. These specimens are deposited in the U.S. National Fungus Collections (BPI), Beltsville, Maryland, United States. Single ascospore or conidial cultures were established as described earlier (Hirooka et al. 2011, 2012). Newly isolated cultures are preserved at the CBS Fungal Biodiversity Centre (CBS, Utrecht, Netherlands), and/or GenBank, National Institute of Agrobiological Sciences (NIAS, Tsukuba, Ibaraki, Japan). Morphology

Methods of morphological observation are described by Hirooka et al. (2011, 2012). To observe sporulating structures, the cultures were grown on a

low nutrient agar (SNA; Nirenberg 1976). Cultures on SNA were incubated at 25℃ with alternating 12h/12h fluorescent light/darkness for 2–3 wks. Images were captured with a Nikon DXM1200 digital camera. Some composite images were made with Helicon Focus version 4.21.5 Pro (Helicon Soft, www.heliconfocus.com). Color terminology used for perithecia, ascospores, conidia, and top and reverse colony, were based on Kornerup & Wanscher (1978). Statistical analysis

Measurements of morphological characters such as length and width were made using Scion Image software beta version 4.0.2 (Scion Corporation, Frederick, Maryland, USA) and are based on up to 50 measurements for structures in each isolate and specimen. For morphological structures, descriptive statistics (minimum, mean, median, maximum and standard deviation) were computed. All computations were performed using Systat 10 (Systat Software, San José, California, USA). Only isolates for which all data existed were included in the analysis. Ranges are reported as mean values ± one standard deviation; the number of items measured is given in parentheses together with maximum and minimum. Growth trial

Disks of 5mm diam. were cut from the edge of young colonies and placed in the center of PDA plates, then incubated at temperatures from 15℃ to 35℃ at 5℃ intervals in complete darkness. Diameters of the colonies on three plates for each isolate at each temperature were measured daily for ten days. This trial was replicated three times for each isolate. DNA extraction, PCR, and sequencing

In total, 75 cultures of Neo. coccinea group and three isolates of Neonectria hederae (C. Booth)


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Castl. & Rossman (Table 1) were grown in Difco™ potato dextrose broth in 6cm diam. Petri plates for about one to two weeks at 20℃. Mycelial mats were obtained in a laminar flow hood and dried with clean, absorbent paper towels. DNA was extracted with Ultra Clean™ Plant DNA Isolation Kit (MO BIO Laboratories Inc., Solana Beach, California, USA).

For each isolate, six loci were sequenced, namely α-actin (act) (Carbone & Kohn 1999), β-tubulin (tub) (O’Donnell & Cigelnik 1997), RNA polymerase II subunit one (rpb1) (Castlebury et al. 2004), the internal transcribed spacer (ITS) (White et al. 1990), large subunit nuclear ribosomal DNA (LSU) (Vilgalys n.d.), and translation elongation factor 1-α (tef1) (Carbone & Kohn 1999; Rehner 2001). The primers and PCR protocol information are listed in Tables 2. PCR products were purified with ExoSAP-IT® (USB Corporation, Cleveland, Ohio, USA) following the manufacturer’s instructions. Clean PCR products were sequenced at the DNA Sequencing Facility (Center for Agricultural Biotechnology, University of Maryland, College Park, Maryland, USA) and at MCLAB (Molecular Cloning Laboratories, San Francisco, California, USA). Sequences were assembled and edited with Sequencher 4.9 (Gene Codes, Madison, Wisconsin, USA). Sequences are deposited in GenBank (Table 1). Phylogenetic analyses

Sequences of the six loci were aligned with MAFFT version 6 (Katoh 2008) and the alignment was visually improved with Mesquite version 2.74 (Maddison & Maddison 2010). Phylogenetic analyses with aligned sequences were performed using the Bayesian (BI), Maximum likelihood (ML) and Maximum parsimony (MP) analyses. Three isolates of Neo. hederae, CBS 714.97, A.R. 4523

and IMI 058770a, were used as the outgroup for inferring inter- and intra-specific relationships.

JMODELTEST (Posada 2008) was used to calculate the models of nucleotide substitutions for each gene/partition for the ML and BI analyses. The number of substitution schemes was set to 11, base frequencies +F, rate variation +I and +G, and the base tree for likelihood calculations was set to ‘ML optimized’. Eighty-eight models were compared. After calculation of likelihood scores, the models were selected according to the Akaike information criterion (AIC) (Posada & Buckley 2004). After jMODELTEST, likelihood settings for trees were set to each gene (Table 2).

BI analysis was done using MrBayes 3.1.2 (Huelsenbeck et al. 2001, 2002). BI data were partitioned by locus and the parameters of the nucleotide substitution models for each partition were set as described (Table 2). For these analyses, two independent analyses of two parallel runs and four chains were carried out for 10,000,000 generations using MrBayes. Analyses were initiated from a random tree and trees sampled every 100th generation. The first 20% of the resulting trees were eliminated (=‘burn in’). A consensus tree (‘sumt’ option) and posterior probabilities (PP) were calculated in MrBayes.

For the ML and bootstrap analyses (BP), GARLI version 0.96 (Zwickl 2006) was computed through the Grid computing (Cummings & Huskamp 2005) and The Lattice Project (Bazinet & Cummings 2008), which includes clusters and desktops in one integrated network (Myers et al. 2008). In GARLI, the starting tree was made by stepwise-addition and the number of runs or search replicates was set to 50. 2000 ML BP replicates were done in GARLI, with the starting tree chosen randomly.



490

Tabl

e 1

Isol

ates

and

acc

essio

n nu

mbe

rs u

sed

in th

e ph

ylog

enet

ic a

naly

ses

Gen

Ban

k ac

cess

ion

No.

Sp

ecie

s Is

olat

e N

o.

Her

bariu

m N

o. S

ubst

rate

/Hos

t C

ount

ry

act

ITS

LSU

rp

b1

tef1

tu

b

Neo

nect

ria

cocc

inea

A

.R. 3

696=

CBS

1195

34

BPI

871

112

Fagu

s syl

vatic

a Fr

ance

K

C66

0420

K

C66

0501

K

C66

0619

K

C66

0670

K

C66

0436

K

C66

0723

Neo

. coc

cine

a A

.R. 3

694=

CBS

1189

14

BPI

870

938

Fagu

s syl

vatic

a Fr

ance

K

C66

0419

K

C66

0500

K

C66

0607

K

C66

0669

K

C66

0435

K

C66

0725

Neo

. coc

cine

a C

BS

1191

58=G

.J.S.

98-

114

BPI

748

295

Fagu

s sp.

G

erm

any

KC

6604

22

KC

6605

21

KC

6606

20

KC

6606

72

JF26

8734

* K

C66

0727

Neo

. coc

cine

a M

AFF

241

561=

TPP-

h430

B

PI 8

8194

2 Fa

gus c

rena

ta

Japa

n K

C66

0426

K

C66

0548

K

C66

0552

K

C66

0674

K

C66

0493

K

C66

0729

Neo

. coc

cine

a A

.R. 3

708=

CBS

1189

16

BPI

870

939

Fagu

s syl

vatic

a R

oman

ia

KC

6604

25

KC

6605

05

KC

6606

01

KC

6606

64

KC

6604

42

KC

6607

19

Neo

. coc

cine

a A

.R. 3

707=

CBS

1189

15

BPI

870

937

Fagu

s syl

vatic

a R

oman

ia

KC

6604

16

- K

C66

0578

K

C66

0666

K

C66

0441

K

C66

0718

Neo

. coc

cine

a G.

J.S. 9

2-33

=CBS

134

254

BPI

802

648

Fagu

s sp.

Sc

otla

nd

KC

6604

18

- K

C66

0581

K

C66

0673

K

C66

0467

K

C66

0722

Neo

. coc

cine

a A

.R. 3

700=

CBS

1191

56

BPI

870

940

Fagu

s syl

vatic

a Sl

ovak

ia

KC

6604

21

- K

C66

0579

K

C66

0671

K

C66

0437

K

C66

0726

Neo

. coc

cine

a A

.R. 3

705=

CBS

1191

50

BPI

870

941

Fagu

s syl

vatic

a Sl

ovak

ia

KC

6604

17

KC

6605

04

KC

6605

77

KC

6606

65

KC

6604

40

KC

6607

17

Neo

. coc

cine

a A

.R. 3

691=

CBS

1195

23

BPI

871

113

Fagu

s syl

vatic

a Sl

ovak

ia

KC

6604

23

KC

6604

99

KC

6606

09

KC

6606

68

KC

6604

34

KC

6607

21

Neo

. coc

cine

a A

.R. 3

712=

CBS

1195

22

BPI

871

115

Fagu

s syl

vatic

a Sl

ovak

ia

KC

6604

24

KC

6605

06

KC

6605

93

KC

6606

67

KC

6604

44

KC

6607

24

Neo

. coc

cine

a A

.R. 3

701

BPI

871

114

Fagu

s syl

vatic

a Sl

ovak

ia

KC

6604

15

KC

6605

02

KC

6605

91

KC

6606

63

KC

6604

38

KC

6607

16

Neo

. coc

cine

a A

.R. 3

687=

CBS

1342

53

- Fa

gus s

ylva

tica

Slov

akia

-

- -

- -

KC

6607

20

Neo

. fag

inat

a=

Cyl

indr

ocar

pon

fagi

natu

m C

BS

217.

67=A

TCC

1654

7=IM

I

1057

38

Fa

gus g

rand

ifolia

Can

ada

KC

6604

12

HQ

8403

85*

HQ

8403

82*

HQ

8403

93*

JF26

8746

* JF

2687

30*

Neo

. fag

inat

a A

.R. 4

307=

CBS

1342

46

BPI

878

329

Fagu

s gra

ndifo

liaU

nite

d St

ates

KC

6604

09

KC

6605

19

KC

6606

00

KC

6606

53

KC

6604

57

KC

6607

43

Neo

. fag

inat

a A

.R. 3

677=

CB

S 11

9154

-

Fagu

s gra

ndifo

liaU

nite

d St

ates

- -

- -

- K

C66

0742

Neo

. fag

inat

a A

.R. 3

682=

CB

S 11

9155

-

Fagu

s gra

ndifo

liaU

n it e

d St

ates

- -

- -

- K

C66

0733

Neo

. fag

inat

a N

CF

346=

AR

368

0=C

BS

1189

83

BPI

880

528

Fagu

s sp.

U

nite

d St

ates

KC

6604

07

KC

6604

98

KC

6605

71

KC

6606

55

KC

6604

33

KC

6607

41

Neo

. fag

inat

a A

.R. 4

130=

CBS

1191

61

BPI

871

095

Fagu

s gra

ndifo

liaU

nite

d St

ates

KC

6604

04

KC

6605

09

KC

6605

57

KC

6606

61

KC

6604

47

KC

6607

36

Neo

. fag

inat

a A

.R. 4

152=

CBS

1189

17

BPI

870

943

Fagu

s gra

ndifo

liaU

nite

d St

ates

KC

6604

08

KC

6605

13

KC

6605

62

KC

6606

58

KC

6604

51

KC

6607

35

Neo

. fag

inat

a A

.R. 4

149=

CBS

1195

19

BPI

871

123

Fagu

s syl

vatic

a U

nite

d St

ates

- K

C66

0511

K

C66

0559

K

C66

0651

K

C66

0449

K

C66

0730

Neo

. fag

inat

a A

.R. 4

197=

CBS

1189

18

BPI

870

945

Fagu

s gra

ndifo

liaU

nite

d St

ates

- K

C66

0518

K

C66

0560

K

C66

0657

K

C66

0456

D

Q78

9856

*

Neo

. fag

inat

a A

.R. 4

167=

CBS

1191

99

BPI

871

125

Fagu

s syl

vatic

a U

nite

d St

ates

KC

6604

06

KC

6605

17

KC

6605

55

KC

6606

52

KC

6604

55

KC

6607

40


Mycosystema

491

Ta

ble

1 co

ntin

ued

Neo

. fag

inat

a A

.R. 4

153=

CBS

1191

63

BPI

871

097

Fagu

s gra

ndifo

liaU

nite

d St

ates

KC

6604

05

KC

6605

14

KC

6605

54

KC

6606

56

KC

6604

52

KC

6607

32

Neo

. fag

inat

a A

.R. 4

151=

CBS

1191

62

BPI

871

096

Fagu

s gra

ndifo

liaU

nite

d St

ates

KC

6604

10

KC

6605

12

KC

6605

72

KC

6606

59

KC

6604

50

KC

6607

37

Neo

. fag

inat

a A

.R. 4

148=

CBS

1195

24

BPI

871

122

Fagu

s syl

vatic

a U

nite

d St

ates

KC

6604

13

KC

6605

10

KC

6606

13

KC

6606

62

KC

6604

48

KC

6607

31

Neo

. fag

inat

a A

.R. 4

097=

CBS

1189

38

BPI

870

942

Fagu

s gra

ndifo

liaU

nite

d St

ates

KC

6604

11

KC

6605

08

KC

6606

14

KC

6606

54

KC

6604

46

KC

6607

39

Neo

. fag

inat

a A

.R. 4

166=

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Not

e: A

.R.:

Am

y Y.

Ros

sman

, USD

A-A

RS

MD

USA

; AT

CC

: A

mer

ican

Typ

e Cu

lture

Col

lect

ion,

Man

assa

s, VA

, USA

; B

PI:

U.S

. Nat

iona

l Fu

ngus

Col

lect

ions

USD

A-A

RS

MD

USA

; C

BS:

Cen

traal

bure

au v

oor

Schi

mm

elcu

lture

s, U

trech

t, Th

e N

ethe

rland

s; C

.L.L

.: Ch

ristia

n Le

chat

, Asc

ofra

nce,

Vill

iers

en

Bois

, Fra

nce.

; G.J

.S.:

Gar

y J.

Sam

uels,

USD

A-A

RS

MD

USA

; J.F

.: Ja

cque

s

Four

nier

, Asc

ofra

nce,

Vill

iers

en

Boi

s, Fr

ance

; MA

FF: M

AFF

Gen

eban

k, N

atio

nal I

nstit

ute

of A

grob

iolo

gica

l Sci

ence

s, Ib

arak

i, Ja

pan;

TU

A-T

PP-h

: Yuu

ri H

irook

a, T

orop

ical

Pla

nt P

lote

ctio

n La

b

Her

bariu

m, T

okyo

Uni

vers

ity o

f A

gric

ultu

re, T

okyo

Jap

an; T

PP-h

: Yuu

ri H

irook

a, T

orop

ical

Pla

nt P

lote

ctio

n La

b Cu

lture

Col

lect

ion,

Tok

yo U

nive

rsity

of

Agr

icul

ture

, Tok

yo J

apan

; Y.H

.: Yu

uri

Hiro

oka,

USD

A-A

RS

MD

USA

. * Sequ

ence

s obt

aine

d fro

m G

enB

ank.



494

Table 2 Parameters used and statistical values resulting from the different phylogenetic analyses of individual datasets for members

of the Neonectria coccinea group

Locus Act ITS LSU Rpb1 Tef1 Tub Combined

Number of

characters

Included sites 663 449 788 650 440 535 3525

Phylogenetically

informative sites (%)

41 (6%) 22 (5%) 11 (1%) 72 (11%) 64 (15%) 64 (12%) 274 (8%)

Tree length 68 47 17 97 135 102 514

Consistency Index (CI) 0.912 0.766 0.941 0.825 0.837 0.863 0.768

Retention Index (RI) 0.978 0.893 0.990 0.968 0.960 0.973 0.942

Rescaled consistency

(RC)

0.892 0.684 0.931 0.798 0.803 0.840 0.724

MP

Homoplasy Index (HI) 0.088 0.234 0.059 0.175 0.163 0.137 0.232

ML and BI Nucleotide substitution

models

TrN+I TPM2uf+I+G TIM3 TrNef+I K80+I TIM1+I

Primers used (reference) Tact1, Tact2

(Samuels et al.

2006)

ITS5, ITS4

(White et al.

1990)

LR5, LROR

(Vilgalys

n.d.)

crpb1a, rpb1c

(Castlebury

et al. 2004)

tef1-728,

tef1-1567

(Carbone &

Kohn 1999;

Rehner 2001)

Btub-T1,

Btub-T2

(O’Donnell

& Cigelnik

1997)

PCR

PCR protocol: annealing

temp. & cycles

65℃, 30s, 15×

48℃, 30s, 30×

53℃, 1min,

35×

53℃, 1min,

35×

50℃, 2min,

40×

66℃, 55s, 9×

56℃, 55s,

35×

55℃, 30s,

35×

MP analyses were performed with PAUP* version 4.0 b10 (Swofford 2002), using 1,000 replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping. Gaps (insertion/deletions) were treated as missing data. Bootstrap analysis was performed with 1,000 replicates, and a 70 % majority rule consensus tree was constructed in PAUP*. The three analyses for each locus, act, tub, rpb1, ITS, LSU, and tef1, were also done and produced each tree to identify the most suitable locus in Neo. coccinea group.

2 RESULTS Phylogenetic analyses

Because the BI, MP and ML trees showed

almost identical topologies in each dataset, the Bayesian trees were taken as representatives (Figs. 1−7). The number of taxa, substitution models and other statistical values resulting from the different analyses are presented in Table 2.

The combined BI, MP, and ML analyses of six loci using 75 isolates of the Neonectria coccinea group resolved four distinct species (Fig. 7). The first major clade (clade A) occurring only in Asia is here determined as Neo. microconidia. This species was supported by moderate values (BI PP 0.73, MP BP 77%, ML BP 74%). In the Neo. microconidia clade, MAFF 241514 and MAFF 241572 formed a clade A-1 supported by high BI PP, MP BP, and ML


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Fig. 1 Bayesian trees with best log likelihoods (Ln−1421.821)

derived from analyses of act data. Thickened branches indicate BI

posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML

bootstrap ≥70%.

BP values. The second major clade (clade B) is recognized as Neo. punicea although this species is supported by less than 0.70 BI PP, 70% MP BP, and 70% ML BP values. Within the Neo. punicea clade, two subclades are highly supported, specifically B-1 with values BI PP 1.00, MP BP 96%, ML BP 94%, and B-2 with values BI PP 1.00, MP BP 100%, ML BP 100%. Neonectria punicea subclade B-1 is known only from Japan while Neo. punicea subclade B-2 is known only from Europe. Neonectria punicea subclade B-2 includes the ex-epitype isolate (A.R. 3102=CBS 119724). The third major clade (clade C) contained two distinct species: Neo. coccinea and Neo. faginata. Neonectria coccinea was supported by moderate BI PP, MP BP, and ML BP values (BI PP 0.86, MP BP 76%, ML BP 75%). At the base of the Neo. coccinea clade (clade E), MAFF 241561 is known only from Asia. The Neo. coccinea subclade E-1 including the ex-type isolate was supported by moderate phylogenetic values (BI PP 0.90, MP BP 75%, ML BP 72%). A fourth species is recognized as Neo. faginata (clade F), previously recognized by Castlebury et al. (2006). Neonectria faginata

occurs only on Fagus in North America. This species is moderately supported (BI PP 0.77, ML BP 72%, ML BP 94%).

The topologies of the individual act, tef1, and rpb1 trees did not contradict each other, and these trees showed the same topologies as the six-locus phylogeny. The topologies of the tub tree showed the

Fig. 2 Bayesian trees with best log likelihoods (Ln–1007.727)

derived from analyses of ITS data. Thickened branches indicate

BI posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML

bootstrap ≥70%.


derived from analyses of LSU data. Thickened branches indicate


bootstrap ≥70%.



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almost similar topologies with the six-locus phylogeny as well as the other protein-coded dataset phylogeny. The topologies of the ITS and LSU trees conflict with the six-locus phylogeny. Among these single locus trees, tef1 provides the best resolution and thus is the most phylogenetically informative.


derived from analyses of rpb1 data. Thickened branches indicate


bootstrap ≥70%.


derived from analyses of tef1 data. Thickened branches indicate


bootstrap ≥70%.


derived from analyses of tub data. Thickened branches indicate BI

posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML

bootstrap ≥70%.

Morphological, colony growth and temperature analyses

The four species in the Neo. coccinea group are difficult to distinguish based solely on teleomorph morphology. Although perithecial characters, such as color, surface, and wall cell structure, are generally reliable for identifying the species or genera of other nectrioid fungi, this is not true for the segregate species of the Neo. coccinea group. The perithecial apex of Neo. coccinea, Neo. faginata, and Neo. punicea tends to be papillate and thus seems to be useful in distinguishing species but this characteristic varies within species. The perithecial wall surface of species in the Neo. coccinea group is slightly roughened. The perithecial walls are about the same thickness and cell walls form similar tissue of textura globulosa to textura angularis; thus, perithecial wall structure is not useful in distinguishing these species. According to Castlebury et al. (2006), the asci of Neo. coccinea are narrowly clavate with an apical ring while those


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Fig. 7 Bayesian tree with best log likelihood (Ln−8382.847) derived from analysis of combined data set (act, ITS, LSU, rpb1, tef1 and tub).

Values at branches indicate Bayesian posterior probabilities (BI PP)/Maximum Likelihood bootstrap (ML BP)/Maximum parsimony

bootstrap (MP BP). BI posterior probabilities ≤0.7, MP bootstrap ≤70%, and ML bootstrap ≤70% indicated by - . Thickened branches

indicate BI posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML bootstrap ≥70%. The culture collection number, geography and host are

included after each taxon name. “EX” indicates ex-type cultures.



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of Neo. faginata are cylindrical without an apical ring and with uniseriate ascospores. In our study, we observed that all species of Neo. coccinea group have uniseriate or occasionally obliquely biseriate ascospores in asci with an apical ring. These morphological characters were not useful in distinguishing species. Although size, surface structure, and septation of ascospores are considered to be important in distinguishing these species, we found no significant differences among the four species of the Neo. coccinea group. We analyzed 95% confidence intervals of length/width ratios of ascospores on natural substrate (Fig. 8). Although length of ascospores in Neo. faginata is statistically shorter than Neo. coccinea, no differences of the length were found among Neo. faginata, Neo. microconidia and Neo. punicea. We were not able to examine the anamorphic states of species in the Neo. coccinea group in nature because we did not find many specimens with the anamorph fructification.

As for the anamorph in culture, colony colors are indistinguishable among species. Colonies are white to umber with ochraceous to sienna reverse. Although the optimal temperature for mycelial growth on PDA for each species is 20℃, the mycelial growth rates at 30℃ for 7days on PDA were different: Neo. punicea and Neo. faginata are less than 15mm diameter while Neo. microconidia and Neo. coccinea are greater than 15mm diameter. The morphology of conidiophores, conidiogenous cells, chlamydospores, and microconidia are similar for the four species of the N. coccinea group; however, these species can be distinguished based on the morphology of the macroconidia. On SNA, Neo. microconidia and Neo. faginata produce up to 8-septate macroconidia while Neo. punicea and Neo. coccinea produce up to 7-septate macroconidia. Size of conidia is also reliable; only Neo. faginata forms

macroconidia that are more than 80μm long when 5-septate while the other species produce macroconidia that are less than 80μm long when 5-septate.

Overall, Neo. faginata is easily identified because it is known only on species of Fagus in North America and produces macroconidia more than 80μm long when 5-septate. Neonectria coccinea is distinguishable based on host specificity occurring only on Fagus in Europe (except for one anomalous isolate from Japan) and the fast mycelial grow at 30℃ for 7d on PDA. Neonectria microconidia and Neo. punicea are similar because of the broad host range and the same size of macroconidia; however, the number of septa in the macroconidia and mycelial growth rate are different. In addition, Neonectria microconidia is known only from Asia. TAXONOMY

Based on our morphological and molecular analyses, the Neonectria coccinea group is re-determined as four distinct species, each of which is described and illustrated below. A key to these four species is provided.

Neonectria coccinea (Pers.: Fr.) Rossman & Samuels, in Rossman, Samuels, Rogerson & Lowen, Stud. Mycol. 42: 158. 1999. Fig. 9

≡Sphaeria coccinea Pers.: Fr., Persoon, Icon. & Descr. Fung. 2: 47. 1800: Fries, Syst. Mycol. 2: 412. 1823.

≡Nectria coccinea (Pers.: Fr.) Fr., Summa Veg. Scand. 2: 388. 1849.

=Fusidium candidum Link, Observationes I, Mag. Ges. Naturf. Freunde Berlin 3: 6. 1809.

≡Cylindrocarpon candidum (Link) Wollenw., Fus. Autogr. Del., ed. 2, no. 655. 1926.

=Sphaerostilbe caespitosa Fuckel, Jahrb. Nassauischen Vereubs Naturk. 27-28: 33. 1873.


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Fig. 8 Graphs of 95% confidence intervals of length to width ratios of ascospores and conidia.

≡Neonectria caespitosa (Fuckel) Wollenw., Angew. Bot. 8: 192. 1926.

=Fusidium fractum Sacc. & Cav., N. Giorn. Bot. ital. 7: 308. 1900.

≡Cylindrocarpon fractum (Sacc. & Cav.) Wollenw., Fus. Autogr. Del., ed. 1, no. 655. 1924.

Teleomorph on natural substrata: Mycelium sometimes visible around perithecia and on host. Stromata up to 0.3mm high and 8mm diam., erumpent through epidermis, red to bay, KOH+ dark red, LA+ yellow, pseudoparenchymatous, cells forming textura angularis to textura prismatica with cells oriented more or less vertically; cells 3–10μm diam., with 1–1.5μm thick walls, intergrading with ascomatal wall. Perithecia superficial on well-developed, erumpent stroma, solitary or

caespitose up to 38 on a stroma, subglobose to pyriform, 218–367μm high and 148–399μm diam. (n=21), red to sienna, often cupulate upon drying or collapsing by lateral pinching, papillate, apical region sometimes slightly darker, KOH+ purple, LA+ yellow, surface with smooth to rough. Perithecial surface cells forming textura globulosa or textura angularis, with walls pigmented, ca. 1.5μm thick. Perithecial wall ca. 17–49μm thick, of two distinct regions: outer region ca. 22–42μm thick, intergrading with stroma, cells forming textura globulosa or textura angularis, walls pigmented, ca. 1.5μm thick; inner region ca. 7–21μm thick, of elongated, thin-walled, hyaline cells, forming textura prismatica. Asci unitunicate, (58–)69–97(–124)× (7.8–)8.9–12.9(–15.9)μm (n=44), cylindrical to



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narrowly clavate, often with an apical ring at apex, stipitate, 8-spored, ascospores occasionally obliquely biseriate near apex. Ascospores ellipsoidal to fusiform with narrowly rounded ends, straight or slightly curved, hyaline, finely spinulose, 1 septate, (9.1–) 11.8–14.6(–19.3)×(3.7–)4.9–6.3(–7.6)μm (n=309).

Anamorph in culture: Optimum temperature for growth on PDA 20℃, colonies 39–56mm (average 45mm) diam. at 20℃ after 7d. Colony surface on PDA, radial, abundant cottony with aerial mycelium, sometimes wavy, white to saffron or umber, saffron to ochraceous sporodochial conidial masses producing after one week; reverse ochraceous to sienna. Sporulation on SNA from lateral phialidic pegs on submerged or commonly on aerial hyphae, 2.0–7.5μm long, 1.5–3.0μm wide at base. Conidiophores occasionally developing on aerial hyphae, unbranched, sometimes verticillate, 1–3-branched, becoming loosely to moderately densely branched, 6.5–124μm long, 2.1–3.0μm wide at base. Conidiogenous cells monophialidic, cylindrical and slightly tapering toward tip or narrowly flask-shaped with widest point in middle, 4–36μm long, 1.5–3.0μm wide at base. Conidia formed by monophialides on submerged or aerial hyphae, formed abundantly on slimy heads, ellipsoidal, oblong to long cylindrical, hyaline, smooth, straight or slightly curved with round at both ends, 0–7-septate; 0-septate: (3.4–)6.0–9.8 (–12.5)×(2.4–)2.7–3.9(–4.6)μm (n=120), 1-septate: 10.0–22.6(–43.5)×(3.6–)3.9–5.1(–5.6)μm (n=52), 2-septate: (22.5–)22.7–29.5(–33.1)×(4.1–)4.4–5.0μm (n=24), 3-septate: (28.1–)32.1–43.5(–52.2)×4.4–6.0 (–7.2)μm (n=48), 4-septate: (41.1–)44.5–62.1 (–62.6)×(5.4–)5.7–6.4(–6.6)μm (n=38), 5-septate: (52.2–)58.9–72.1(–80.4)×(4.2–)5.5–67.1(–8.0)μm (n=114), 6-septate: (70.0–)73.6–84.2(–85.7)×(5.3–) 5.3–6.3(–6.4)μm (n=18), 7-septate: (72.9–)88.4–

100.8(–111.0)×(5.1–)5.4–6.2(–6.5)μm (n=17). Chlamydospores intercalary in hyphae, globose to subglobose, sometimes ellipsoidal, smooth, hyaline. Perithecia not produced in culture.

Distribution: Asia (Japan) and Europe (France, Germany, Romania, Scotland, Slovakia).

Habitat: On Fagus crenata, F. sylvatica and Fagus sp.

Lectotype of Neonectria coccinea designated by Booth (1959). Herb. Lugd. Bat. No. 910, 270-56 on bark of Fagus, isolectotype BPI 738862.

Epitype of Neonectria coccinea designated here. Germany Saarland, Naturpark Saar-Hundsruck, between Leisel and Schollen, alt. 600m. 49°40'0''N 7°10'0''E, on hardwood, Fagus sp., Oct. 13, 1998, coll. G.J. Samuels, H.J. Schroers (BPI 748295; ex-epitype culture CBS 119158=G.J.S. 98-114).

Additional type specimens: A holotype of Sphaeria caespitosa exists at G, isotype K, and BR, according to Booth (1966) and Seifert (1985).

Additional specimens and isolates examined: France, on Fagus sylvatica, 7 Sep. 2001, coll. A. Kunca (BPI 871112; culture A.R. 3696=CBS 119534); on Fagus sylvatica, 7 Sep. 2001, coll. A. Kunca (BPI 870938; culture A.R. 3694=CBS 118914). Japan, Kanagawa Prefecture, Ashigarakami-gun, on Fagus crenata, 17 Apr. 2005, coll. H. Masuya, Y. Hirooka (BPI 881942; culture MAFF 241561=TPP-h430). Romania, Brasov, Busteni, on Fagus sylvatica, 26 Sep. 2000, coll. A. Kunca (BPI 870939; culture A.R. 3708=CBS 118916); Brasov, Busteni, on Fagus sylvatica, 26 Sep. 2000, coll. A. Kunca (BPI 870937; culture A.R. 3707=CBS 118915). Scotland, Cowal Peninsula, Argyll Forest Park. N. End of Loch Goil, alt. 25–250m, on Fagus sp., 12 Apr. 1992, coll. G.J. Samuels, D. Brayford (BPI 802648; culture G.J.S. 92-33=CBS 134254). Slovakia, Kacin, Male Karpety,


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Fig. 9 Neonectria coccinea. A, B: Perithecia in the natural environment; C: Median section of perithecium; D: Median section of perithecial

apex and wall; E: Ascus apices; F: Asci; G: Ascospores in optical section; H: Ascospores in surface view; I: Colony of A.R. 3700 after 7d at

20℃ on PDA; J: Colony of A.R. 3691 after 7d at 20℃ on PDA; K: Colony of A.R. 3707 after 7d at 20℃ on PDA; L: Colony of G.J.S.

92-33after 7d at 20℃ on PDA; M, N, Q–S: Conidiophores on SNA; O, P: Lateral phialidic pegs; T–V: Micro- and macroconidia on SNA.

Bar: A=3mm; B=300µm; C, M=100µm; D=30µm; E–H, N=20µm; I–L=30mm; O–V=10µm.



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on Fagus sylvatica, 14 Apr. 2000, coll. A. Kunca (BPI 870940; culture A.R. 3700=CBS 119156); Muranky planina, Pod Vel’kow stozkou, on Fagus sylvatica, 26 Oct. 2000, coll. A. Kunca (BPI 870941; culture A.R. 3705=CBS 119150); Bansky Studenec 5, Stiavnicke vrchy, on Fagus sylvatica, 10 Jun. 2001, coll. A. Kunca (BPI 871113; culture A.R. 3691=CBS 119523); Bansky Studenec 1, on Fagus sylvatica, 14 Mar. 2001, coll. A. Kunca (A.R. 3687=CBS 134253), Vel’ke Rovne, Javornicky, on Fagus sylvatica, 11 Jul. 2001, coll. A. Kunca (BPI 871115; culture A.R. 3712=CBS 119522); Nova Bystrica, Kysucke vrchy, on Fagus sylvatica, 20 Nov. 2000, coll. A. Kunca (BPI 871114; A.R. 3701).

Notes: Neonectria coccinea is here determined to include specimens collected on Fagus spp. from Europe and one isolate from Japan. To distinguish this species from others in the Neo. coccinea group, cultural characters are required. Neonectria coccinea is similar to Neo. microconidia in having 5-septate conidia that are 50–70μm long on SNA and a growth rate in culture of more than 15mm at 30℃ for 7days on PDA. In addition, septation of macroconidia in Neo. coccinea ranges from 1–7-septate while in Neo. microconidia the range is 1–8-septate. Also, Neo. coccinea is known only from Fagus while Neo. microconidia occurs on Fagus and other hosts. Surprisingly our phylogenetic analyses suggest a sister-group relationship between Neo. coccinea and MAFF 241561 collected from Japan (Fig. 7). However, based on our observations of the morphological characters in the natural environment and culture, MAFF 241561 completely matches Neo. coccinea. Although there are obvious geographical differences, we tentatively regard MAFF 241561 as Neo. coccinea.

Castlebury et al. (2006) mentioned that BPI 748295 and the derived CBS 119158 as a

representative isolate of N. coccinea but an epitype was not designated. Since this species is economically important, an epitype of the basionym Sphaeria coccinea is needed to distinguish Neo. coccinea from other species in the Neo. coccinea group. The specimen (BPI 748295, ex-epitype culture CBS 119158) is herein designate as epitype of S. coccinea. This specimen was collected on Fagus sp. from Germany, most likely the type locality of Sphaeria coccinea collected by Persoon (1800) because Persoon moved to Göttingen for botanical research and earned a doctorate from the Academy of Natural Sciences in Erlangen in 1777 (de Zeeuw 1939; Petersen 1977).

Neonectria faginata (M.L. Lohman, A.M.J. Watson & Ayers) Castl. & Rossman, in Castlebury, Rossman & Hyten, Can. J. Bot. 84: 1425. 2006. Fig. 10

≡Neonectria coccinea var. faginata M.L. Lohman, A.M.J. Watson & Ayers, Lloydia 6: 100. 1943.

=Cylindrocarpon faginatum C. Booth, Mycol. Pap. 104: 1. 1966.

Teleomorph on natural substrata: Mycelium sometimes visible around perithecia and on host. Stromata up to 0.3mm high and 8mm diam., erumpent through epidermis, red to bay, KOH+ dark red, LA+ yellow, pseudoparenchymatous, cells forming textura angularis to textura prismatica with cells oriented more or less vertically; cells 3–9μm diam., with 1–2μm thick walls, intergrading with ascomatal wall. Perithecia superficial on well-developed, erumpent stroma, solitary or caespitose up to 50 on a stroma, subglobose to pyriform, 195–399μm high and 156–302μm diam. (n=45), red to bay, often cupulate upon drying or collapsing by lateral pinching, papillate, apical region sometimes slightly darker, KOH+ purple,


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LA+ yellow, surface with smooth to rough. Perithecial surface cells forming textura globulosa or textura angularis, with walls pigmented, ca. 1.5μm thick. Perithecial wall ca. 31–50μm thick, of two distinct regions: outer region ca. 17–42μm thick, intergrading with stroma, cells forming textura globulosa or textura angularis, walls pigmented, ca. 1.5μm thick; inner region ca. 13–20μm thick, of elongated, thin-walled, hyaline cells, forming textura prismatica. Asci unitunicate, (65.8–)69.4–96.4 (–124.5)×(7.0–)8.9–11.9(–13.7)μm (n=47), cylindrical to narrowly clavate, often with an apical ring at apex, stipitate, 8-spored, ascospores occasionally obliquely biseriate near apex. Ascospores ellipsoidal to fusiform with narrowly rounded ends, straight or slightly curved, hyaline, finely spinulose, 1-septate, (8.5–)10.3–12.3(–14.3)×(4.1–)4.4–6.2 (–8.0)μm (n=249).

Anamorph in culture: Optimum temperature for growth on PDA 20℃, colonies 34–40mm (average 36mm) diam. at 20℃ after 7d. Colony surface on PDA, radial, abundant cottony with aerial mycelium, sometimes wavy, ochraceous, saffron to ochraceous sporodochial conidial masses producing after one week; reverse ochraceous. Sporulation on SNA from lateral phialidic pegs on submerged or commonly on aerial hyphae, 2.0–6.8μm long, 1.5–2.5μm wide at base. Conidiophores occasionally developing on aerial hyphae, unbranched, sometimes verticillate, 1–3-branched, becoming loosely to moderately densely branched, 6.5–87μm long, 2.1–3.0μm wide at base. Conidiogenous cells, monophialidic, cylindrical and slightly tapering toward tip or narrowly flask-shaped with widest point in middle, 8–29μm long, 1.5–2.5μm wide at base. Conidia formed by monophialides on submerged or aerial hyphae, formed abundantly on slimy heads, ellipsoidal, oblong to long cylindrical, hyaline,

smooth, straight or slightly curved with round at both ends, 0–8-septate; 0-septate: (2.5–)7.5–11.9 (–13.2)×(2.8–)3.3–4.5(–4.8)μm (n=140), 1-septate: (13.2–)13.4–16.6(–19.0)×(3.6–)3.7–4.7(–5.0)μm (n=40), 2-septate: (13.7–)15.5–25.9(–30.3)×(3.1–) 4.0–5.1(–5.8)μm (n=25), 3-septate: (20.4–)35.8– 50.5(–55.6)×(4.5–)4.8–6.7(–7.0)μm (n=21), 4-septate: (44.4–)59.1–67.2(–73.4)×(4.4–)4.8–6.2(–6.8)μm (n=22), 5-septate: (75.9–)76.7–92.3(–97.1)×(5.6–) 5.8–6.8(–7.0)μm (n=50), 6-septate: (78.2–)79.5– 92.1(–104.5)×(5.1–)5.9–7.2(–8.0)μm (n=14), 7-septate: (93.1–)94.1–102.3(–102.7)×5.0–6.6(–7.7)μm (n=35), 8-septate: (95.5–)97.3–109.0(–114.1)×(5.1–)5.9–7.5 (–8.1)μm (n=20). Chlamydospores intercalary in hyphae, globose to subglobose, sometimes ellipsoidal, smooth, hyaline. Perithecia not produced in culture.

Distribution: North America (Canada, United States).

Habitat: On Fagus grandifolia, F. sylvatica and Fagus sp.

Holotype of Neonectria faginata: United States, Maine, Meddybemps, on Fagus grandifolia, 4 Oct. 1933, coll. T.T. Ayers (BPI 551558).

Epitype of Neonectria faginata designated here: United States, Maine, on Fagus grandifolia, Apr. 2006, coll. M. Kassen (BPI 878329; ex-epitype culture A.R. 4307=CBS 134246).

Ex-holotype culture of Cylindrocarpon faginatum: Canada, New Brunswick York County, Rocky Brook, on Fagus grandifolia (CBS 217.67=ATCC 16547=IMI 105738).

Specimens and isolates examined: United States, New Hampshire, Grafton Co., Hubbard Brook Experimental Forest, on Fagus grandifolia, 1998, coll. E.M. Mahoney (culture A.R. 3677=CBS 119154); Maine, Penobscot Co., Penobscot Experimental Forest, on Fagus grandifolia, 1998,



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Fig. 10 Neonectria faginata. A–C: Perithecia in the natural environment; D: Median section of perithecium; E: Median section of perithecial

apex and wall; F: Asci; G: Ascospores in optical section; H: Ascospores in surface view; I: Colony of A.R. 4167 after 7d at 20℃ on PDA; J:

Colony of A.R. 4307 after 7d at 20℃ on PDA; K–O, Q–U: Conidiophores on SNA; M: Lateral phialidic pegs and conidiophores on SNA; P,

V: Micro- and macroconidia on SNA; W: Chlamydospores on SNA. Bar: A=3mm; B, C=300µm; D=100µm; E, F, K=30µm; G, H,

L–W=10µm; I, J=30mm.


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coll. E.M. Mahoney (culture A.R. 3682=CBS 119155); West Virginia, on Fagus sp., coll. E.M. Mahoney (BPI 880528; culture NCF 346=AR 3680=CBS 118983); West Virginia. Burner Mt. BBD plot tree 4, on Fagus grandifolia, Oct. 2004, coll. M. MacKenzie (BPI 871095; culture A.R. 4130=CBS 119161; West Virginia, Burner Mt. BBD plot tree 8, on Fagus grandifolia, Mar. 03 2005, coll. M. MacKenzie (BPI 870943; culture A.R. 4152=CBS 118917; North Carolina, Blue Ridge Parkway National Park, Richland Balsam, on Fagus sylvatica, Mar. 25 2004, coll. M. MacKenzie (BPI 871123; culture A.R. 4149=CBS 119519); Michigan, Luce Co., N of Bass Lake on County Road 421. 46.5°N 85.78°W, on Fagus grandifolia, Jun. 29 2005, coll. G. Joseph O’Brien (BPI 870945; culture A.R. 4197=CBS 118918); Pennsylvania, McKean Co., Bradford Rd., on Fagus sylvatica, May 12 2005, coll. M. MacKenzie (BPI 871125; culture A.R. 4167=CBS 119199); West Virginia, Burner Mt. BBD plot, tree gamma, on Fagus grandifolia, Mar. 31 2005, coll. M. MacKenzie (BPI 871097; culture A.R. 4153=CBS 119163; West Virginia, Burner Mt. BBD plot, tree beta, on Fagus grandifolia, Mar. 31 2004, coll. M. MacKenzie (BPI 871096; culture A.R. 4151=CBS 119162; North Carolina, Great Smoky Mountains National Park. Newfound Gap, on Fagus sylvatica, Dec. 03 2003, coll. B. Jones (BPI 871122; culture A.R. 4148=CBS 119524); Pennsylvania, Allegheny National Forest, tree 53, on Fagus grandifolia, Oct. 07 2004, coll. M. MacKenzie (BPI 870942; culture A.R. 4097=CBS 118938; West Virginia, Monogahela National Forest. Cheat Mt. Road, on Fagus sylvatica, May 04 2005, coll. M. MacKenzie (BPI 871124; culture A.R. 4166=CBS 119198).

Notes: Neonectria faginata was raised to the rank of species by Castlebury et al. (2006) based on

morphology, geography and phylogeny. Among the four species in the Neo. coccinea group, the morphology of Neo. faginata is relatively distinctive. The five-septate macroconidia of Neo. faginata are longer than the other species i.e., 80–90μm long in Neo. faginata vs. 50–70μm long in other species of Neo. coccinea group. Neonectria faginata is known only on Fagus spp. in North America. According to Castlebury et al. (2006), only Fagus grandifolia was recorded as the host of Neo. faginata. Four specimens on Fagus sylvatica are reported here.

The holotype specimen of Neo. coccinea var. faginata is preserved in BPI, but it lacks a living ex-type culture. Therefore, we epitypify this name with BPI 878329 (ex-epitype culture A.R. 4307=CBS 134246) collected from the same host and state in the United States as the holotype.

Neonectria microconidia J. Luo, P. Zhao & W.Y. Zhuang, in Zhao, Luo, Zhuang, Liu & Wu, Sci. China Life Sci. 54: 671. 2011. Fig. 11

Teleomorph on natural substrata: Mycelium sometimes visible around perithecia and on host. Stromata up to 0.4mm high and 12mm diam., erumpent through epidermis, red to bay, KOH+ dark red, LA+ yellow, pseudoparenchymatous, cells forming textura angularis to textura prismatica with cells oriented more or less vertically; cells 3–12μm diam., with 1–2μm thick walls, intergrading with ascomatal wall. Perithecia superficial on well-developed, erumpent stroma, solitary or caespitose up to 300 on a stroma, subglobose to pyriform, 203–380μm high and 175–357μm diam. (n=30), red to sienna, often cupulate upon drying or collapsing by lateral pinching, sometimes papillate, apical region sometimes slightly darker, KOH+ purple, LA+ yellow, surface with smooth to rough. Perithecial surface cells forming textura globulosa



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or textura angularis, with walls pigmented, ca. 1.5μm thick. Perithecial wall ca. 30–44μm thick, of two distinct regions: outer region ca. 20–40μm thick, intergrading with stroma, cells forming textura globulosa or textura angularis, walls pigmented, ca. 1.5μm thick; inner region ca. 10–18μm thick, of elongated, thin-walled, hyaline cells, forming textura prismatica. Asci unitunicate, (58–)64–94 (–102)×(6.3–)8.0–10.6(–11.1)μm (n=35), cylindrical to narrowly clavate, often with an apical ring at apex, stipitate, 8-spored, ascospores occasionally obliquely biseriate near apex. Ascospores ellipsoidal to fusiform with narrowly rounded ends, straight or slightly curved, hyaline, finely spinulose, 1-septate, (8.9–)10.6–15.8(–23.1)×(3.8–)4.5–6.5(–9.4)μm (n=192).

Anamorph in culture: Optimum temperature for growth on PDA 20℃, colonies 34–56mm (average 47mm) diam at 20℃ after 7d. Colony surface on PDA, radial, abundant cottony with aerial mycelium, sometimes wavy, white to ochraceous, saffron to ochraceous sporodochial conidial masses producing after one week; reverse ochraceous to rust. Sporulation on SNA from lateral phialidic pegs on submerged or aerial hyphae common, 2.0–7.2μm long, 1.5–2.5μm wide at base. Conidiophores occasionally developing on aerial hyphae, unbranched, sometimes verticillate, 1–3-branched, becoming loosely to moderately densely branched, 6.5–110μm long, 2.1–3.0μm wide at base. Conidiogenous cells, monophialidic, cylindrical and slightly tapering toward tip or narrowly flask-shaped with widest point in middle, 6–38μm long, 1.5–2.5μm wide at base. Conidia formed by monophialides on submerged or aerial hyphae, formed abundantly on slimy heads, ellipsoidal, oblong to long cylindrical, hyaline, smooth, straight or slightly curved with round at both ends,

0–8-septate; 0-septate: (3.1–)5.5–8.9(–12.3)×(1.7–) 2.1–3.3(–4.9)μm (n=288), 1-septate: (9.8–)12.2–19.2 (–31.0)×(2.5–)3.5–4.5(–5.6)μm (n=144), 2-septate: (12.2–)16.7–28.5(–34.8)×(3.4–)4.2–5.4(–5.6)μm (n=37), 3-septate: (24.6–)31.5–43.9(–49.7)×(4.0–) 4.8–6.2(–7.1)μm (n=84), 4-septate: (39.9–)44.5– 53.3(–57.7)×(4.4–)4.8–6.2(–6.8)μm (n=42), 5-septate: (47.4–)55.7–69.7(–76.4)×(4.0–)5.5–6.9(–7.6)μm (n=95), 6-septate: (63.9–)69.3–88.7(–101.9)×(5.0–) 5.9–7.5(–8.2)μm (n=54), 7-septate: (71.5–)80.3– 98.3(–118.2)×(5.1–)6.2–7.6(–8.5)μm (n=51), 8-septate: (79.1–)85.2–101.7(–120.0)×(5.2–)6.6–7.2(–8.0)μm (n=17). Chlamydospores intercalary in hyphae, globose to subglobose, sometimes ellipsoidal, smooth, hyaline. Perithecia not produced in culture.

Distribution: Asia (China, Japan). Habitat: On dead woody substrata, known from

Cerasus jamasakura, Fagus crenata, Vitis coignetiae and Weigela coraeensis.

Holotype of Neonectria microconidia: China, Hubei, Wufeng, 800m alt., on twigs, 13 Sep. 2004, coll. W.Y. Zhuang, Y. Nong (5639) (HMAS 98294).

Specimens and isolates examined: Japan, Fukuoka Prefecture, Oazanatuki, Tagawa city, on bark of dead wood, Apr. 02 2004, coll. Y. Hirooka (BPI 882098; culture MAFF 241530=TPP-h260); Kanagawa Prefecture, Yamakawa-cho, Ashigarakami-gun, on bark of dead wood, Oct. 31 2004, coll. Y. Hirooka (BPI 881931; culture MAFF 241556=TPP-h385); Kanagawa Prefecture, Kiyokawa-mura, Aigo-gun, on twigs, Apr. 16 2005, coll. Y. Hirooka (BPI 881941; culture MAFF 241560=TPP-h427); Kanagawa Prefecture, Yamakawa-cho, Ashigarakami-gun, on dead wood of Cerasus jamasakura, Oct. 31 2004, coll. Y. Hirooka (BPI 881933, culture MAFF 241558=TPP-h390), Kanagawa Prefecuutre, Atugi city, on stump, May 03 2003, coll. Y. Hirooka (culture MAFF


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241506=TPP-h120); Kanagawa Prefecture, Yamakitagawayose, Ashigarakami-gun, on Fagus crenata, Jul. 03 2005, coll. Y. Hirooka (BPI 881956; culture MAFF 241570=TPP-h521); Kanagawa Prefecture, Yamakawa-cho, Ashigarakami-gun, Genbu river, on bark of dead wood, Oct. 31 2004, coll. Y. Hirooka (BPI 881934, culture MAFF 241559=TPP-h391); Kanagawa Prefecture. Yamakawa-cho, Ashigarakami-gun, on bark of dead wood, Oct. 31 2004, coll. Y. Hirooka (BPI 881930; culture MAFF 241555=TPP-h378); Kochi Prefecture, Tosakitakaido, Tosa-cho, on twigs, Aug. 04 2003, coll. Y. Hirooka (BPI 882163; culture MAFF 241516=TPP-h176); Kochi Prefecture, Shirono, Kitagawa-mura, on twigs, Aug. 05 2003, coll. Y. Hirooka (BPI 882165; culture MAFF 241518=TPP-h190); Nagano Prefecture, Sugadaira, Ueda city, on twigs of Vitis coignetiae, Sep. 01 2006, coll. Y. Hirooka (BPI 881958; culture MAFF 241572=TPP-h542); Saitama Prefecture, Nakaizu, Tagata-gun, on twigs of Weigela coraeensis, Oct. 14 2002, coll. Y. Hirooka (BPI 882137; culture MAFF 241493=TPP-h64); Tokyo, Okutama-gun, on twigs, Nov. 20 2003, coll. Y. Hirooka (culture MAFF 241522=TPP-h227); Yamagata Prefecture, Akakura, mogami-cho, on bark of dead wood, Jul. 26 2003, coll. T. Tokiwa (BPI 882161; culture MAFF 241514=TPP-h171); Yamanashi Prefecture, Yamato-son, Higashiyamanashi-gun, Yamato-son, on stem, Sep 26, 2009, coll. T. Tokiwa, Y. Hirooka (BPI 882119; culture MAFF 241552=TPP-h341).

Notes: Neonectria microconidia was recently described from China (Zhao et al. 2011). Based on our phylogenetic analyses, the type isolate of this species clustered with many Japanese isolates. This clade is supported by more than 0.70 MI PP, 70% MPBP and 70% ML BP values (clade A in Fig. 7). Within the Neo. microconidia clade, subclade A-1 is

supported by high BIPP, MPBP and MLBP values. Although subclade A-1 includes only Japanese isolates MAFF 241514 and MAFF 241572, no morphological differences were found from the other isolates of Neo. microconidia. Thus subclade A-1 is not considered to be distinct from Neo. microconidia.

Neonectria microconidia is known only from Asia although Neo. punicea and one isolate of N. coccinea are also known from Asia. Neonectria microconidia can be distinguished from the other species of the Neo. coccina group using the morphological characters of the anamorph. Neonectria microconidia has 5-septate macroconidia that are 50–70μm long, conidia varying from 1–8-septate, a colony growth rate of more than 15mm in diameter after 7d at 30℃ and a wide host range.

Neonectria punicea (J.C. Schmidt: Fr.) Castl. & Rossman, in Castlebury, Rossman & Hyten, Can. J. Bot. 84: 1425. 2006. Fig. 12

≡Sphaeria punicea J.C. Schmidt, in Kunze & Schmidt, Mykologische Hefte (Leipzig) 1: 61. 1817.

=Fusarium album Sacc., Michelia 2: 132. 1880. ≡Cylindrocarpon album (Sacc.) Wollenw.,

Fusaria autographica delineate 1: no. 473. 1916. =Neonectria confusa J. Luo & W.Y. Zhuang,

Mycologia 102: 143. 2010. Teleomorph on natural substrata: Mycelium

sometimes visible around perithecia and on host. Stromata up to 0.4mm high and 10mm diam., erumpent through epidermis, scarlet to bay, KOH+ dark red, LA+ yellow, pseudoparenchymatous, cells forming textura angularis to textura prismatica with cells oriented more or less vertically; cells 4–14μm diam., with 1–1.5μm thick walls, intergrading with ascomatal wall. Perithecia superficial on well-



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Fig. 11 Neonectria microconidia. A–C: Perithecia in the natural environment; D: Median section of perithecium; E: Median section of

perithecial wall; F, G: Asci; H: Ascospores in optical section; I: Ascospores in surface view; J: Colony of MAFF 241516 after 7d at 20℃ on

PDA; K: Colony of MAFF 241493 after 7d at 20℃ on PDA; L: Colony of MAFF 241530 after 7d at 20℃ on PDA; M: Colony of MAFF

241559 after 7d at 20℃ on PDA; N, P–W: Conidiophores on SNA; O: Lateral phialidic pegs on SNA; X: Micro- and macroconidia on SNA.

Bar: A=3mm; B, C=300µm; D=100µm; E, U, V=30µm; F, G=20µm; H, I=5µm; J–M=30mm; N–T, W, X=10µm.


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developed, erumpent stroma, solitary or caespitose up to 40 on a stroma, subglobose to pyriform, 211–364μm high and 160–338μm diam. (n=50), red to sienna, often cupulate upon drying, papillate, apical region slightly darker, KOH+ purple, LA+ yellow, surface with smooth to rough. Perithecial surface cells forming textura globulosa or textura angularis, with walls pigmented, ca. 1.5μm thick. Perithecial wall ca. 17–46μm thick, of two distinct regions: outer region ca. 16–34μm thick, intergrading with stroma, cells forming textura globulosa or textura angularis, walls pigmented, ca. 1.5μm thick; inner region ca. 6–15μm thick, of elongated, thin-walled, hyaline cells, forming textura prismatica. Asci unitunicate, (48–)69–93(–109)× (6.1–)8.1–11.3(–14)μm (n=100), cylindrical to narrowly clavate, often with an apical ring at apex, stipitate, 8-spored, ascospores occasionally obliquely biseriate near apex. Ascospores ellipsoidal to fusiform, sometimes long fusiform, with narrowly rounded ends, straight or slightly curved, hyaline, finely spinulose, 1-septate, (8.3–)11.0–14.4(–18.5)× (2.9–)4.2–5.8(–7.6)μm (n=675).

Anamorph in culture: Optimum temperature for growth on PDA 20℃, colonies 31–54mm (average 41mm) diam at 20℃ after 7d. Colony surface on PDA, radial, abundant cottony with aerial mycelium, sometimes wavy, white to saffron or ochraceous, saffron to ochraceous sporodochial conidial masses producing after one week; reverse ochraceous to sienna. Sporulation on SNA from lateral phialidic pegs on submerged or aerial hyphae common, 2.0–7.5μm long, 1.5–3.0μm wide at base. Conidiophores occasionally developing on aerial hyphae, unbranched, sometimes verticillate, 1–3-branched, becoming loosely to moderately densely branched, 6.5–98μm long, 2.1–3.5μm wide at base. Conidiogenous cells, monophialidic,

cylindrical and slightly tapering toward tip or narrowly flask-shaped with widest point in middle, 4–24μm long, 2.5–3.5μm wide at base. Conidia formed by monophialides on submerged or aerial hyphae, formed abundantly on slimy heads, ellipsoidal, oblong to long cylindrical, hyaline, smooth, straight or slightly curved with round at both ends, 0–7-septate: 0-septate: (3.2–)6.1–9.7 (–18.3)×(1.5–)2.3–3.7(–6.1)μm (n=505), 1-septate: (8.6–)11.8–20.6(–27.7)×(2.3–)3.6–5.0(–6.6)μm (n=241), 2-septate: (20.3–)22.7–28.7(–31.2)×(4.0–) 4.3–6.1(–7.0)μm (n=50), 3-septate: (25–)31.4–42.2 (–52.5)×(3.5–)4.5–6.1(–8.0)μm (n=233), 4-septate: (37.7–)43.0–54.6(–64.9)×(4.3–)4.9–6.1(–7.2)μm (n=94), 5-septate: (40.9–)51.6–67.9(–79.9)×(4.0–) 5.2–6.6(–8.0)μm (n=199), 6-septate: (66.3–)68.6– 79.2(–84.7)× (4.7–)5.2–7.0(–7.8)μm (n=25), 7-septate: (80.7–)81.9–102.1(–103.8)×(5.0–)5.2–6.8(–6.9)μm (n=21). Chlamydospores intercalary in hyphae, globose to subglobose, sometimes ellipsoidal, smooth, hyaline. Perithecia not produced in culture.

Distribution: Asia (China, Japan); Europe (Austria, France, Germany, Scotland, Slovakia, Switzerland); North America (United States).

Habitat: On dead woody substrata including Acer macrophyllum, Acer sp., Frangula alnus, Fagus grandifolia, F. sylvatica, Prunus×yedoensis, Quercus crispula, Rhamnus fallax, Rhamnus sp., and Ulmus sp.

Neotype of Sphaeria punicea designated here: Austria, Kaernten, St. Margareten i. Ros., "Tumpfi". Grid square 9452/4, on Frangula alnus, 26 Sep. 1999, W. Jaklitsch (BPI 871063; ex-neotype culture A.R. 3102=CBS 119724).

Ex-type of Neonectria confuse: China, Hubei, Wufeng, 1,200m, on twigs of a dicotyledonous tree, 15 Sep. 2004, coll. Y. Nong, W.Y. Zhuang (Y. Nong, W.Y. Zhuang 5694; HMAS 99197).



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Specimens and isolates examined: Austria, Kaernten, Bad Vellach, Vellacher Kotschna. MTB 8653/1, on Rhamnus fallax, 17 Aug. 2004, coll. W. Jaklitsch (BPI 871062; culture A.R. 4155=CBS 119527). France, Pyrenees Atlantiques, Isle de Sauveterre de Bearn, alt. 100m, on hardwood tree, 25 Oct. 1998, coll. G.J. Samuels, F. Candoussau (BPI 748311; culture G.J.S. 98-133=CBS 134255); Foret du St. Sauvant, on dead twigs of Frangula alnus, 18 Apr. 2008, coll. A. Rossman (BPI 878875; culture A.R. 4522=CBS 134247); Village de Combret, 48 St. Germain du Teil, on Castanea, 19 Feb. 2011, coll. C. Lechat C.L.L. 10040 (A.R. 4828=CBS 134248); St. Maixent, on Robinia, 14 Feb. 2011, coll. C. Lechat C.L.L. 10042 (A.R. 4829=CBS 134249); Lauastric, on Salix, 12 Feb. 2011, coll. C. Lechat C.L.L. 10045 (A.R. 4830=CBS 134250); Las Muros, 09 Rimont, on Acer, 14 Mar 2011, coll. J. Fournier J.F. 11017 (A.R. 4831=CBS 134251); La Maille, 09 Rimont, on Salix, 25 May 2011, on Salix, 25 Mar 2011, coll. J. Fournier J.F. 11023 (A.R. 4832=CBS 134252). Germany, on bark, Mar. 1924, coll. H.W. Wollenweber (BPI 871038; culture CBS 125.24=IMI 113880=MUCL 9808); on Ulmus sp., Jul. 1930, coll. H.W. Wollenweber (BPI 871035=Centraalbureau voor Schimmelcultures Herbarium H-11427; culture CBS 208.30); former West-Germany, on bark of Rhamnus sp., Jul. 1929, coll. H.W. Wollenweber (culture CBS 242.29). Japan, Gifu Prefecture, Mizunashi city, Kashimo-mura, on twigs, 31 Aug. 2004, coll. Y. Hirooka (BPI 882115; culture MAFF 241548= TPP-h328); Niigata Prefecture Nakauonuma-gun, Tuna-cho, Akiyamago, on twigs of Prunus× yedoensis, 30 Aug. 2004, coll. Y. Hirooka (BPI 882113; culture MAFF 241546=TPP-h326); Gifu Prefecture, Mizunashi city, Kashimo-mura, on twigs,

31 Aug. 2004, coll. Y. Hirooka (BPI 882117; culture MAFF 241550=TPP-h330); Tokyo, Okutama-gun, on bark of dead wood, 12 Jul. 2003, coll. T. Tokiwa (BPI 882160; culture MAFF 241513=TPP-h166); Miyagi Prefecture, Taihaku-ku, akiu-cho, Akiuootaki, on twigs, 4 Aug. 2004, coll. Y. Hirooka (culture MAFF 241540=TPP-h296); Niigata Prefecture, Nakauonuma-gun, Tuna-cho, on bark, 31 Aug. 2004, coll. Y. Hirooka (BPI 882114; culture MAFF 241547=TPP-h327); Miyagi Prefecture, Taihaku-ku, akiu-cho, Akiuootaki, on twigs, 4 Aug. 2004, coll. Y. Hirooka (BPI 882107; culture MAFF 241541=TPP-h297); Miyagi Prefecture, Taihaku-ku, akiu-cho, Akiuootaki, on Quercus crispula, 4 Aug. 2004, coll. Y. Hirooka (BPI 882104; culture MAFF 241537=TPP-h290); Gifu Prefecture, Mizunashi city, Kashimo-mura, on twigs, 31 Aug. 2004, coll. Y. Hirooka (BPI 882116; culture MAFF 241549=TPP-h329). Scotland, Cowal Peninsula, Argyll Forest Park. N. End of Holy Loch, alt. 25–250m, on dead bark of Acer macrophyllum, 13 Apr. 1992, coll. G.J. Samuels, D. Brayford (BPI 802647; culture G.J.S. 92-32=CBS 119530). Slovakia, Volvec, Volovske vrchy, on Fagus sylvatica, 29 Oct. 1999, coll. A. Kunca (BPI 871118; culture A.R. 3713=CBS 119531); Stagiar, on Fagus sylvatica, 1999, coll. I. Mihal, A. Kunca (BPI 871116; A.R. 3454=CBS 119525); Stagiar, Kremnicke vrchy, on Fagus sylvatica, 16 Jul. 1999, coll. A. Kunca (BPI 871117; A.R. 3711=CBS 119533); Poruba-N6, on Fagus sylvatica, coll. A. Kunca (culture A.R. 3704=CBS 119532). Switzerland, Grisons (Graubunden), Canton, Malan Vicinity, alt. ca. 600m, on Fagus sylvatica, 5 Sep. 1990, coll. K.F. Rodrigues (BPI 1107108; culture G.J.S. 90-29=CBS 119529). United States, New York, Ulster Co., Catskill Mts. from Oliverea at end


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Fig. 12 Neonectria punicea. A–C: Perithecia in the natural environment; D, E: Median section of perithecia; F: Asci; G: Ascospores in

surface view; H: Colony of MAFF 241513 after 7d at 20℃ on PDA; I: Colony of MAFF 241549 after 7d at 20℃ on PDA; J: Colony of

CBS 125.24 after 7d at 20℃ on PDA; K, M–U: Conidiophores on SNA; L: Lateral phialidic pegs on SNA; V, W: Micro- and macroconidia

on SNA; X: Chlamydospores on SNA. Bar: A=3mm; B–D=300µm; E, K, R=100µm; F=20µm; G, L–N=5µm; H–J=30mm; O–Q,

S–X=10µm.



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of McKinley Hollow Road to Summit of Balsam Mt., alt. 550–1,098m. 42°4'0''N 74°22'0''W, on Acer sp., 7 May 1993, coll. W.R. Buck (BPI 802504; culture G.J.S. 93-35=CBS 119528); New York, Schuyler Co., Arnot Forest, on Fagus grandifolia,1988, coll. E.M. Mahoney (culture A.R. 3675=CBS 119153); Pennsylvania, Allegheny National Forest. Tree 6, on Fagus grandifolia, 7 Oct. 2004, coll. M. MacKenzie (BPI 870944; culture A.R. 4096=CBS 119231).

Notes: The basionym of Neonectria punicea, Sphaeria punicea, was described from Germany in 1817 (Kunze & Schmidt 1817), and the type specimens should be preserved in B. Despite intensively searching in several herbaria including B around Europe, this type specimen could not be located. Because no illustrations exist in the original paper (Kunze & Schmidt 1817), the name is herein

neotypified. The one isolate from Germany on Rhamnus sp., the original host of Sphaeria punicea, did not have a teleomorph specimen. A specimen with culture from Austria on Frangula alnus (previously known as Rhamnus frangula; Rhamnaceae) is herein designated the neotype of Sphaeria punicea (BPI 871063; ex-neotype culture A.R. 3102=CBS 119724).

Morphologically, Neo. punicea could be difficult to identify because it has a wide host range and is known in Asia, Europe, and North America. Based on our phylogenetic data, at least two cryptic species are embedded within Neo. punicea. These appear as subclades B-1 and B-2 each supported by high MI PP, MPBP and ML BP values. Although these specimens and isolates of these subclades were examined carefully, we could not find useful characters to distinguish them.

Key to species in the Neonectria coccinea GROUP

1. Mycelial growth >15mm at 30℃ for 7d on PDA············································································ 2

1. Mycelial growth <15mm at 30℃ for 7d on PDA············································································ 3

2. Macroconidia 1−7-septate; on Fagus; in Europe and Asia (one isolate from Japan) ····Neo. coccinea

2. Macroconidia 1−8-septate; known only from Asia (China and Japan)··················Neo. microconidia

3. Macroconidia 1−7-septate, 5-septate macroconidia 50–70µm long on SNA; known from diverse

woody hosts in Asia (China and Japan), Europe, and North America ··························· Neo. punicea

3. Macroconidia 1−8-septate, 5-septate macroconidia 80–90µm long on SNA; known only on Fagus

in North America·········································································································Neo. faginata

3 DISCUSSION Species delimitation

By combining a morphological species concept (John & Maggs 1997; Kirk et al. 2008) with genealogical concordance phylogenetic species recognition (Taylor et al. 2000), we are able to define four species in the Neonectria coccinea group. Similarly this approach has been used to divide the Nectria cinnabarina complex into four

species (Hirooka et al. 2011). In the N. cinnabarina species complex anamorphic states in nature showed the most useful characters for defining species. In the Neo. coccinea group these characters were rarely found.

Our phenotypic and genotypic analyses of the Neo. coccinea group delimited four previously known species, each of which is described and illustrated. Based on our analyses as well as the


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phylogenetic tree in Zhao et al. (2011), Neo. coccinea, Neo. faginata, Neo. microconidia and Neo. punicea form a monophyletic group within the genus Neonectria sensu Chaverri (Chaverri et al. 2011). Of the six loci used, two nuclear ribosomal DNA regions, ITS and LSU datasets, did not fully distinguish these species, but sequence data from the four protein-coding genes, act, tef1, tub, and rpb1, strongly supported them. In the tub dataset one isolate of Neo. punicea, A.R. 4155=CBS 119527, was not nested within the main Neo. punicea clade due to transitions of a few base pairs. However, this isolate falls in the Neo. punicea clade in the phylogeny produced by the other three protein-coding genes as well as in the combined dataset. This phylogenetic separation resulting from a few base pair positions also occurred in the Grosmannia serpens complex (Duong et al. 2012). Among the protein-coding genes used in this study, the tef1 dataset provided the most variable regions and thus was the most informative. Sequence data of the ITS, the official barcoding gene for fungi (Seifert 2009; Schoch et al. 2012), did not reliably resolve relationships within the Neo. coccinea group.

In our study, Neo. faginata and Neo. coccinea were segregated based on phylogenetic and morphological data as mentioned in Castlebury et al. (2006). Within Neo. punicea many distinct clades are included, although only two of them are phylogenetically supported by more than 0.70 MI PP, 70% MP BP and 70% ML BP values. The ex-neotype culture of the Neo. puniciea designated herein as A.R. 3102 (CBS 119724) is included in Neo. punicea subclade B-2 together with CBS 242.29 and A.R. 4522 (=CBS 134247) supported by high phylogenetic values. These three isolates could be regarded as Neo. punicea sensu stricto; the other isolates of Neo. punicea could be taxonomically

segregated from the sensu stricto isolates. Likewise, subclade B-1 of Neo. punicea consisting of isolates only from Japan is supported by high MI PP, MP BP and ML BP values. However, because no morphological differences could be observed, we do not recognize these two clades as distinct species. Similar subclades exist in Neo. coccinea and Neo. microconidia, but again no morphological characters exist on which to base species delimitations. Geography

The Neonectria coccinea group is known from throughout the world especially in the Northern Hemisphere (Castlebury et al. 2006). Within each species, geography is more or less useful. Neonectria microconidia is known only in Asia, Neo. faginata only in North America, and Nectria coccinea without MAFF 241561 only from Europe. Neonectria punicea is widespread in Asia, Europe and North America. These four species have been reported in tropical regions and the Southern Hemisphere (Doidge 1950); however, none of these reports could be confirmed. Host

Known only on Fagus, Neonectria coccinea and Neo. faginata are recognized as the causal agents of beech bark disease in Europe and North America, respectively. On the other hand Neo. punicea and Neo. microconidia are collected from a wide range of woody shrubs and trees including Fagus. Although we have not examined the pathogenicity of Neo. microconidia and Neo. punicea, they could be plant pathogenic. Most specimens used in this study were collected on newly killed branches or trunks. These fungi may exist as endophytes and then sporulate when the substrate becomes weakened (Chapela & Boddy 1988, Sieber 2007).

Knowledge of the geographic distribution and



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host association of species in the Neo. coccinea group has been limited primarily to North America and Europe. In our study, many samples of Neo. coccinea group from Asia, especially Japan, were included; most of these are not species of Neo. coccinea and Neo. faginata, cause of BBD. Surprisingly, MAFF 241561 from Fagus crenata in Kanagawa, Japan, appears to be basal to the rest of the isolates of Neo. coccinea on Fagus in Europe (subclade E-1). Subclade E-1 including the ex-type culture (CBS 119158) was supported by moderate to high MI PP, MP BP and ML BP values (BI PP 0.90, MP BP 75%, ML BP 72%) values. We propose two hypotheses about the origin of MAFF 241561. First MAFF 241561 may have been introduced from Europe to Japan and thus derived from European Neo. coccinea by a mutation. This is suggested by the high number of shared single nucleotide polymorphisms, the sister relationship between MAFF 241561 and the E-1 subclade indicates a recent speciation event. The host of MAFF 241561 is Japanese beech, Fagus crenata, a species that is sister to the European beech, Fagus sylvatica (Denk 2003). Another hypothesis is that MAFF 241561 may have originated in Japan and simply was not detected until now. To know the origin of the Japanese isolate, more collections are needed not only from Japan, but also from other Asian countries. The potential threat of BBD in Japan

Non-indigenous fungal pathogens can be highly disastrous to hosts that are closely related to the original host species because of host-pathogen co-adaptation (Parker & Gilbert 2004). The current forest inventory data in the United State suggest that BBD has invaded areas with relatively high densities of beech. BBD have degraded American beech trees (Fagus grandifolia) in the eastern deciduous forests of North America and are now spreading in

Canadian forests (Houston 1994; O’Brien et al. 2001; MacKenzie & Iskra 2005).

In our study, MAFF 241561 is identified for the first time as Neo. coccinea in Japan based on phenotypic and genotypic data. This fungus may already have spread around Japan and will cause serious damage to Japanese beech trees sooner or later as has occurred in North America. So far, the disease caused by this fungus has not been detected in Japan, possibly because the beech bark scale (Cryptococcus fagisuga) has not colonized Japan (Gwiazdowski et al. 2006). The scale insect makes feeding wounds in the bark that enables infection by Neonectria spp. In order to determine and prevent the possible threat of BBD, Japanese beech trees should be tested for pathogenicity using MAFF241561. A careful watch for the beech bark scale insect as well as other isolates of Neo. coccinea in Japan should be continued.

Acknowledgements: The authors thank Dr. Gary J.

Samuels (USDA-ARS, Beltsville, Maryland, USA) for

using the many specimens and isolates that he collected as

well as providing fruitful comments during the course of

this study. We also thank the many mycologists who

shared their fresh specimens and/or cultures with us,

specifically Matt Kassen, Walter Jaklitsch, Christian

Lechat, Martin MacKenzie, Hayato Masuya, and

Toshiyuki Tokiwa. We express sincere thanks to Sato

Toyozo, Takayuki Aoki, and Keisuke Tomioka (NIAS

Genebank, National Institute of Agrobiological Sciences,

Tukuba, Ibaraki, Japan), and Keiko T. Natuaki (Tokyo

University of Agriculture) for quickly depositing and

sending Japanese cultures. We express sincere thanks to

the curators and staff of the BPI and G. This study was

supported in part by the United States National Science

Foundation (NSF) PEET grant DEB-0731510

‘Monographic Studies in the Nectriaceae, Hypocreales:


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